1. Field of the Invention
The present invention relates to a method for the quantitative release of natural or recombinant proteins, polypeptides or peptides (thermostable immunoligands) able to bind to the Fc-part of immunoglobulins (antibodies, in particular of the IgG class and primarily becoming bound outside the paratope) from complexes in various sample matrixes in order to make these released natural or recombinant proteins, polypeptides or peptides quantitatively available in immunochemical assays and to keep them quantitatively available. The method of release is primarily intended to be used as a prestep in the immunoassay of the kind of proteins, polypeptides and peptides just mentioned when they exist as contaminants in various immunoglobulin/antibody preparations or as more or less pure preparations.
In the context of the present application, the term xe2x80x9cof bacterial originxe2x80x9d refers to a polypeptide or protein which is, derived from polypeptide or protein that is produced naturally by bacteria or other microorganisms. xe2x80x9cRecombinantxe2x80x9d designates a protein, a polypeptide or a shorter antibody binding fragment expressed in any type of cells by means of genetic engineering. xe2x80x9cSynthetic peptidexe2x80x9d means that the part of a protein or polypeptide which contains the functional region is produced by means of chemical peptide synthesis.
2. Description of Related Art
Naturally occurring proteins that are able to bind to the Fc part of primarily class G immunoglobulins (Ig) (IgG) of most mammals are mainly of bacterial origin. The binding strength differs according to species and subclass. This property was first described regarding protein A (SPA) of staphylococcus aureus in 1966 (Forsgren A., Sjxc3x6quist J. J. Immunol. 1966;17:822-27) and, a little later, regarding protein G (SPG) of streptococcus pneumoniae in 1973 (Kronvall G., J. Immunol. 1973;111:1401-06). These proteins bind to antibodies in the region between the constant domains 2 and 3 (CH2 and CH3) of the heavy chains. Proteins A and G are known to be able to bind additionally to the variable region of the heavy chains of IgA, IgE, IgG and IgM, which Igs belong to the VH3 family (Inganxc3xa4s M. et al., Scand. J. Immunol. 1981; 14:379-88 and Erntell M. et al., Scand. J. Immunol. 1983; 17:201-09). Amino acids of three sections in the variable region of the heavy chains of the antibodies (FR1, CDR2 and FR3) are involved in this so-called xe2x80x9calternative reactivityxe2x80x9d (Potter K N. et al., J. Immunol. 1996;157(7):2982-88 and Potter K N. et al., Int. Rev. Immunol. 1997;14(4):291-308). Proteins A and G bind antibodies either at the same site or, at least, at closely overlapping sites (Eliasson M. et al., J. Immunol. 1989;142(2):575-81). Protein A has 5 (A-E) IgG binding subunits (Moks T. et al., Eur.J.Biochem. 1986; 153(3):637-43). The xe2x80x9calternative reactivityxe2x80x9d may be a function of all-single domains, however, it has successfully been examined for two fragments (domains), for domain B (Inganxc3xa4s M. et al., Scand.J.Immunol. 1980;12:23-31, Inganxc3xa4s M. et al., Scand.J.Immunol. 1981;13:343-352, Inganxc3xa4s M. et al., Scand.J.Immunol. 1981;14:379-388) and for domain D (Roben P W. et al., J. Immunol. 1995;154(12):6437-45).
Apart from proteins A and G, further antibody binding proteins are known, which, however, have different specificities and binding regions, such as, e.g., protein H (Akesson P. et al., Mol. Immunol. 1990;27(6):523-31) or clusterine (Wilson M R. et al., Biochim. Biophys. Acta 1992; 1159(3):319-326). The above mentioned IgG binding proteins, polypeptides and peptides (binding outside the paratope) are relatively resistant to heat treatment (in solution) and will therefore in the present document be referred to as thermostable immunoligands.
These proteins (particularly protein A) are used in a variety of applications and are employed on a large scale mainly in the purification of monoclonal and polyclonal antibodies. Both the native and recombinasnt forms are used as ligands in immunoaffinity chromatography. This refined technology provides highly effective purification of antibodies from complex solutions. The antibodies are usually bound at moderate pH to a chromatographic matrix carrying as ligand one of these immunoglobulin-binding proteins and desorbed in an acid environment (pH=2.7-3.5, or 2.7-3.2). Under these conditions, however, it has been impossible so far to avoid a certain degree of ligand leakage.
This is of major importance for antibody preparations used for clinical applications. Protein A (the same applies to protein G) is assumed to have high biological activity, and many publications describe toxic effects in animal models and in humans (Bensinger W I. et al., J. Biol. Response Mod. 1984; 3 (3):347-51, Messerschmidt G L. et al., J. Biol. Response Mod. 1984;3(3):325-29, Terman D S. et al., Eur. J. Cancer Clin. Oncol. October 1985; 21(10):1115-22, Ventura G J. et al., Cancer Treat. Rep. April 1987; 71(4):411-13). Along with enterotoxines A and B, protein A is also thought to play a role in the pathogenicity of staphylococcus aureus infections. Due to its xe2x80x9calternative reactivityxe2x80x9d, it can also cause mitogenic stimulation of family VH3 B-cells. Therefore, it is essential that these ligands can be identified sensitively, specifically and, above all, correctly, in immunoglobulin preparations.
Many publications and review articles deal with possible ways to use polypeptides able to bind to the Fc-part of antibodies, and with their potential dangers when present as contaminants in products for clinical applications (e.g. Langone J J. et al., J. Adv. Immunol. 1982;17:157-252).
So far, immunochemical assays, such as ELISA (Enzyme Linked Immuno Sorbent Assay) or RIA (Radio Immuno Assay) have been employed in different variations to detect and quantify Ig-binding proteins and/or polypeptides.
One problem with this type of assays is that an Fc(IgG)-binding protein (analyte) will form complexes with the Fc part of antibodies/immunoglobulin G with different affinity depending on the species, IgG subclass and even antibody. This has previously been solved by using either antibody fragments (Fab, Fab2xe2x80x2) or specific chicken antibodies (which are not bound by proteins A or G at the Fc-part) for detection (e.g. Langone J J. et al., J. Immunol. Meth. 1982;63:145-57).
In order to release Fc(IgG)-binding proteins and polypeptides from the Fc part of IgG in order to identify them in the presence of IgG, the samples have conventionally been assayed in an acid environment (pH=3.2 or 3.5, respectively) (Berglund A. and Inganxc3xa4s M., U.S. Pat. No. 4,752,571, 1988; Knicker S. et al., J. Immunol. Meth. 1991; 142:53-59). This method is useful, for instance, to determine protein A in samples containing mouse IgG (various subclasses) at a concentration of up to 250 xcexcg of IgG/ml.
This previously known method has the following limitations:
1. Not all antibody-ligand complexes can be broken up under the conditions used previously. The efficiency of this method depends on the species, the amount of antibodies and the sample matrix. This method is particularly unsuitable for human IgG and plasma and serum samples.
2. It is not possible to further reduce the pH, as would be necessary in many cases, because this would also prevent the reaction with the antibodies used in the assay.
3. Acidification of the samples leads to protein precipitation of various intensity depending on the sample matrix, which causes variable degrees of loss of the proteins or peptides to be detected.
4. Slight deviations in pH lead to different reaction behaviour in the assay and may cause the standard protein dilutions and sample dilutions to react with different intensity in the assay.
It has been suggested to use a combination of SDS (sodium dodecyl hydrogensulfate) and DETAPAC (diethylene triamine-pentaacetic acid) combined with a heating step in order-to make immune complexed antigen molecules (bound by the Fab parts of antibodies) available for immunoassays (AT 403,378 A1, Steindl F.). The release of the antigen depends on the fact that the treatment renders complexing via the antigen binding part (Fab) of the antibody difficult. However, binding at the Fc-Part of IgG is something else than Fab-binding.
Accordingly, for the detection in general of proteins or polypeptides (thermostable immunoligands) recognizing only regions in individual chains of antibodies, however, this method is unsuitable for the following reasons:
1. The renaturation of the heavy chains,in the Fc region and thus the re-binding of such proteins and polypeptides to the heavy chains is not prevented in an optimal way.
2. Various molecules used as buffer ions in affinity chromatography, particularly those containing amino groups (amino acids, e.g. glycine, histidine, or molecules, such as TRIS) also adsorb SDS during heat treatment and thus reduce efficiency. These effects make it hard to adapt the SDS concentration to the protein concentration, particularly in samples having a low protein content.
3. The reagent solution used has only very little buffer capacity (regarding pH). As this method has been optimized primarily for plasma and serum samples, optimum adjustment of the SDS concentration requires previous knowledge of the samples (protein content, type of buffer ions in the sample). This means that the method is not generally applicable. In the case of aunknown samples, it is usually impossible to find the optimum SDS concentration at the first attempt.
4. The period of heat treatment is too short and the SDS concentration is not high enough to optimally denaturate the tertiary structure of antibodies and thus of the specific antigen binding regions (paratopes) and to maintain quantitative dissociation in the sample dilutions for the assay.
In view of the needs of the prior art, the present invention provides a method for releasing thermostable immunoligands that are bound to the Fe-part of immunoglobulins in immunoglobulin-immunoligand complexes. The method includes the step of mixing a sample containing said immunoglobulin-immunoligand complexes with a reagent compound that binds non-specifically or adsorbs to said immunoglobulins and heating said sample mixed with reagent compound and thereafter cooling said sample mixed with reagent compound.